DNA Methylome Regulates Virulence and Metabolism in Pseudomonas syringae

Reviewer #1 (Public Review):

Summary:

In this work, Huang et al used SMRT sequencing to identify methylated nucleotides (6mA, 4mC, and 5mC) in Pseudomonas syringae genome. They show that the most abundant modification is 6mA and they identify the enzymes required for this modification as when they mutate HsdMSR they observe a decrease of 6mA. Interestingly, the mutant also displays phenotypes of change in pathogenicity, biofilm formation, and translation activity due to a change in gene expression likely linked to the loss of 6mA.

Overall, the paper represents an interesting set of new data that can bring forward the field of DNA modification in bacteria.

Major Concerns:

• Most of the authors' data concern Psph pathovar. I am not sure that the authors' conclusions are supported by the two other pathovars they used in the initial 2 figures. If the authors want to broaden their conclusions to Pseudomonas synringae and not restrict it to Psph, the authors should have stronger methylation data using replicates. Additionally, they should discuss why Pss is so different than Pst and Psph. Could they do a blot to confirm it is really the case and not a sequencing artefact? Is the change of methylation during bacterial growth conserved between the pathovar? The authors should obtain mutants in the other pathovar to see if they have the same phenotype. The authors have a nice set of data concerning Psph but the broadening of the results to other pathovar requires further investigation.

• The authors should include proper statistical analysis of their data. A lot of terms are descriptive but not supported by a deeper analysis to sustain the conclusions. For example, in Figure 4E, we do not know if the overlap is significant or not. Are DEGs more overlapping to 6mA sites than non-DEGs? Here is a non-exhaustive list of terms that need to be supported by statistics: different level (L145), greater conservation (L162), significant conservation (L165), considerable similarity (L175), credible motifs (L189), Less strong (L277) and several "lower" and "higher" throughout the text.

• The authors performed SMRT sequencing of the delta hsdMSR showing a reduction of 6mA. Could they include a description of their results similar to Figures 1-2. How reduced is the 6mA level? Is it everywhere in the genome? Does it affect other methylation marks? This analysis would strengthen their conclusions.

• In Figure 6E to conclude that methylation is required on both strands, the authors are missing the control CAGCN6CGC construct otherwise the effect could be linked to the A on the complementary strand.

Reviewer #2 (Public Review):

In the present manuscript, Huang et.al. revealed the significant roles of the DNA methylome in regulating virulence and metabolism within Pseudomonas syringae, with a particular focus on the HsdMSR system in this model strain. The authors used SMRT-seq to profile the DNA methylation patterns (6mA, 5mC, and 4mC) in three P. syringae strains (Psph, Pss, and Psa) and displayed the conservation among them. They further identified the type I restriction-modification system (HsdMSR) in P. syringae, including its specific motif sequence. The HsdMAR participated in the process of metabolism and virulence (T3SS & Biofilm formation), as demonstrated through RNA-seq analyses. Additionally, the authors revealed the mechanisms of the transcriptional regulation by 6mA. Strictly from the point of view of the interest of the question and the work carried out, this is a worthy and timely study that uses third-generation sequencing technology to characterize the DNA methylation in P. syringae. The experimental approaches were solid, and the results obtained were interesting and provided new information on how epigenetics influences the transcription in P. syringae. The conclusions of this paper are mostly well supported by data, but some aspects of data analysis and discussion need to be clarified and extended.

Reviewer #3 (Public Review):

Summary:

The article by Huang et.al. presents an in-depth study on the role of DNA methylation in regulating virulence and metabolism in Pseudomonas syringae, a model phytopathogenic bacterium. This comprehensive research utilized single-molecule real-time (SMRT) sequencing to profile the DNA methylation landscape across three model pathovars of P. syringae, identifying significant epigenetic mechanisms through the Type-I restriction-modification system (HsdMSR), which includes a conserved sequence motif associated with N6-methyladenine (6mA). The study provides novel insights into the epigenetic mechanisms of P. syringae, expanding the understanding of bacterial pathogenicity and adaptation. The use of SMRT sequencing for methylome profiling, coupled with transcriptomic analysis and in vivo validation, establishes a robust evidence base for the findings

Strengths:

The results are presented clearly, with well-organized figures and tables that effectively illustrate the study's findings.

Weaknesses:

It would be helpful to add more details, especially in the methods, which make it easy to evaluate and enhance the manuscript's reproducibility.